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  • C12P13/24—Proline; Hydroxyproline; Histidine

Definitions

• This invention relates to a process for producing L-phenylalanine by a novel method for the expression of a gene. More specifically, the present invention relates to a process for producing L-phenylalanine by transforming a host microorganism belonging to the genus Corynebacterium or Brevibacteriou with a recombinant DNA of a DNA fragment containing a gene involved in the biosynthesis of L-phenylalanine and a vector DNA, culturing the transformant in a nutrient medium, accumulating the L-phenyl­alanine in the culture medium and recovering it therefrom.
• the present invention relates to the production of L-phenylalanine using a microorganism belonging to the genus Corynebacterium or Brevibacterium by recombinant DNA technology different from the conventional mutational breeding technology for the purpose of improving the amino acid productivity.
• the present inventors have constructed plasmid vectors autonomously replicable in a microorganism belonging to the genus Corynebacterium or Brevibacterium and having selectable markers and adequate cloning sites and have developed a highly efficient transformation system (Japanese Published Unexamined Patent Application Nos. 183799/82, 186492/82 and 186489/82).
• the present inventors have found that the plasmid vectors are useful for expressing a foreign gene in a host microorganism and increasing the productivity of amino acids by ligating a DNA fragment containing a foreign gene involved in the biosynthesis of amino acids such as glutamic acid and lysine to the plasmid vectors according to the procedures in recombinant DNA technology (Methods in Enzymology 68 , Recombinant DNA, edited by Ray Wu, Academic Press 1979, U.S. Patent No. 4,237,224) and transforming Corynebacterium glutamicum L-22 or its derivatives using the transformation methods developed by the present inventors (Japanese Published Unexamined Patent Application No. 126789/83).
• the present inventors have found that microorganisms prepared by the above method acquire an increased productivity of phenylalanine.
• the present invention provides a process for producing L-phenylalanine by cultivating in a medium a transformant which is obtained by transforming a microorganism belonging to the genus Corynebacterium or Brevibacterium with a recombinant DNA of a DNA fragment containing a gene involved in the biosynthesis of L-phenylalanine and a vector DNA.
• the DNA fragment containing the gene used in the present invention the DNA fragment containing a gene involved in the biosynthesis of L-phenylalanine derived from eukaryotes, prokaryotes, viruses, bacteriophages or plasmids is used.
• the gene derived from prokaryotes the gene derived from a bacterium belonging to the genus Escherichia , Corynebacterium , Brevibacterium , Microbacterium , Bacillus , Staphylococcus , Streptococcus or Serratia and involved in the biosynthesis of L-phenylalanine of the present invention or the metabolism relating to the biosynthesis is preferably used.
• amino acid-­producing strains and amino acid analog-resistant strains derived from the bacteria described above are used as the most preferable source of the gene.
• the amino acid analog-resistance can be conferred on a plasmid after cloning.
• the genes coding for chorismate mutase and prephenate dehydratase are mentioned. Further, the above-mentioned genes on which the phenotype resistant to phenylalanine, tyrosine or an analog thereof such as para­fluorophenylalanine (PFP) is conferred are used. As the source of such a gene, Corynebacterium glutamicum K38 resistant to PFP is mentioned.
• Metabolic pathway and regulation systems of aromatic amino acids in microorganisms have been studied in detail on Escherichia coli , Bacillus subtilis , and glutamic acid-producing microorganisms such as strains of the genus of Corynebacterium and Brevibacterium [Agr. Chem. Soc. Japan, 50 (1), R79 - R87 (1976) and Ann. Rev. Biochemistry 47 , 533 (1978)].
• the gene involved in the biosynthesis of tyrosine is a DNA carrying a genetic information of at least one of the enzymes involved directly or indirectly in the biosynthesis of these aromatic amino acids.
• DAHP 3-deoxy-D-arabino-heptulosonate 7-phosphate
• CMase chorismate mutase
• PDGase prephenate dehydrogenase
• the vector used in the present invention should autonomously replicate in cells of the host microorganism.
• plasmids isolated from microorganisms belonging to the genus Corynebacterium by the present inventors or derivatives thereof such as pCG1 (Japanese Published Unexamined Patent Application No. 134500/82), pCG2 (Japanese Published Unexamined Patent Application No. 35197/83, pCG4 (Japanese Published Unexamined Patent Application No. 183799/82), pCE51, pCE52 (Japanese Published Unexamined Patent Application No. 126789/83), pCE53 (Japanese Published Unexamined Patent Application No. 25398/83), pCE54, pCG11, pCB100 (Japanese Published Unexamined Patent Application No. 105999/83), and the like are mentioned,
• Plasmids pCE51, 52, 53 and 54 can be introduced from the plasmids mentioned above as follows.
• pCE51 is prepared as follows.
• pCG1 is isolated from the cultured cells of Corynebacterium glutamicum 225-57 (FERM P-5865, ATCC 31808) by the method described in the specification of Japanese Published Unexamined Patent Application No. 134500/82.
• pGA22 is isolated from the cultured cells of Escherichia coli harboring the plasmid by a conventional method [An, G. et al .,: J. Bacteriol., 140 , 400 (1979)].
• Plasmid pCG1 is linearized with restriction endonuclease BglII and a fragment of pGA22 digested with BamHI and containing kanamycin resistance (Km R ) gene is ligated to the linearized pCG1 using the same cohesive ends of both plasmids. Isolation of pCE51 from the ligated DNA mixture is achieved by selecting the transformants belonging to the genus Corynebacterium or Brevibacterium and containing KM R derived from pGA22, and analyzing the plasmid in the transformant.
• pCE51 has a molecular weight of about 6 Kb and cleavage sites for HincII, HindIII, SmaI, XhoI and EcoRI and gives Km R phenotype.
• pCE52 and pCE53 are prepared as follows.
• Plasmid pCG1 is isolated from the cultured cells of Corynebacterium glutamicum 225-57 (FERM P-5865, ATCC 31808) by the method described in the above application and plasmid pGA22 is isolated from the cultured cells of Escherichia coli harboring the plasmid by a conventional method.
• pCG1 with a unique BglII site is linearized with restriction enzyme BglII and pGA22 with two BamHI sites are partially digested with BamHI. The cohesive ends of both plasmids are annealed and ligated with T4 phage DNA ligase to make a composite molecule.
• Selection of the recombinant plasmids from the ligation mixture is carried out by isolating transformants of the genus Corynebacterium or Brevibacterium on the basis of drug-­resistances derived from pGA22 and then analyzing the plasmids in the transformants.
• pCE52 and pCE53 have a molecular weight of about 10.9 Kb and cleavage sites for EcoRI, SalI, SmaI and XhoI. While pCE52 gives the phenotypes of resistance to chloramphenicol (Cm R ) and Km R , pCE53 gives resistance to tetracycline (Tc R ), Cm R and Km R phenotypes. Since the cleavage site for XhoI is present in the Km R gene, selection by insertional inactivation (prevention of the expression of a gene by the insertion of a DNA fragment into the gene) is possible.
• Transformation with the ligated DNA mixture is carried out using protoplasts of the genus Corynebacterium or Brevibacterium , and the method described in Japanese Published Unexamined Patent Application Nos. 186492/82 and 186489/82.
• transformants are recovered as a colony regenerated on a hypertonic agar medium containing generally 0.4 - 1.6 ⁇ g/ml Tc, 2.5 - 5 ⁇ g/ml Cm, 100 - 800 ⁇ g/ml Km, 100 - 400 ⁇ g/ml Sm or 200 - 1,000 ⁇ g/ml Spec which does not allow the reversion to normal cells of the recipient protoplasts which are not treated with the ligation mixture.
• transformants are regenerated unselectively on a regeneration medium, and the resultant cells are scraped and resuspended, followed by the isolation of those cells grown on an agar medium containing a drug in a concentration wherein thr recipient normal cells can not grow, that is, generally, 0.5 - 4 ⁇ g/ml Tc, 2 - 15 ⁇ g/ml Cm, 2 - 25 ⁇ g/ml Km, 5 - 50 ⁇ g/ml Sm or 50 - 500 ⁇ g/ml Spec.
• Some of the transformants selected by Tc R , Cm R or Km R are simultaneously endowed with other drug-­resistances derived from plasmid pGA22.
• Plasmid DNAs in these transformants can be isolated from cultured cells of the transformants and purified according to the methods described in Japanese Published Unexamined Patent Application Nos. 134500/82 and 186489/82.
• the structures of the DNAs can be determined by digesting them with various restriction endonucleases and analyzing the DNA fragments by agarose gel electrophoresis.
• the plasmids isolated from the transformants are pCE51, pCE52 and pCE53.
• Recovery of plasmids from the strains is carried out according to the methods described in Japanese Published Unexamined Patent Application Nos. 134500/82, 183799/82 and 35197/83.
• Preparation of a recombinant DNA of a vector DNA with a DNA fragment containing a gene is carried out by conventional in vitro recombinant DNA technology.
• the ligase reaction gives recombinants containing genes other than the desired gene.
• the desired recombinant DNA can be obtained by directly transforming a microorganism of the genus Corynebacterium or Brevibacterium with the ligated DNA mixture, selecting the transformants having the phenotype derived from the desired gene and isolating the desired recombinant DNA from the cultured cells of the transformants.
• the desired gene can be cloned by using another host-vector system such as Escherichia coli . Then, it is recloned in vitro into a vector of the genus Corynebacterium or Brevibacterium to transform these microorganisms and transformants containing the desired recombinant plasmid are selected as mentioned above.
• Microorganisms belonging to the genus Corynebacterium or Brevibacterium and which are competent for incorporating DNAs may be used as the host microorganisms in the present invention.
• Transformation of the host microorganisms with recombinant DNAs is carried out by the following steps:
• the preparation of protoplasts is carried out by culturing a microorganism under conditions which render it sensitive to lysozyme, a lytic enzyme, and treating the cultured cells with lysozyme in a hypertonic solution to remove the cell wall.
• a microorganism under conditions which render it sensitive to lysozyme, a lytic enzyme, and treating the cultured cells with lysozyme in a hypertonic solution to remove the cell wall.
• reagents inhibiting the synthesis of bacterial cell walls are used.
• microbial cells sensitive to lysozyme are obtained by adding, during the logarithmic growth phase, an amount of penicillin which does not inhibit or sub-inhibits the growth and then continuing culturing for several generations.
• any medium wherein the microorganism can grow may be used.
• a nutrient medium NB (pH 7.2) consisting of 20 g/l powdered bouillon and 5 g/l yeast extract and a semi-synthetic medium SSM (pH 7.2) consisting of 10 g/l glucose, 4 g/l NH4Cl, 2 g/l urea, 1 g/l yeast extract, 1 g/l KH2PO4, 3 g/l K2HPO4, 0.4 g/l MgCl2 ⁇ 6H2O, 10 mg/l FeSO4 ⁇ 7H2O, 0.2 mg/l MnSO4 ⁇ (4-6)H2O, 0.9 mg/l ZnSO4 ⁇ 7H2O, 0.4 mg/l CuSO4 ⁇ 5H2O, 0.09 mg/l Na2B4O7 ⁇ 10H2O, 0.04 mg/l (NH4)6MO7O24 ⁇ 4H2O, 30 ⁇ g/l bio
• Microbial cells are inoculated in the medium and culturing is carried out with shaking.
• the optical density (OD) of the culture medium at 660 nm is monitored with a colorimeter and penicillin, such as penicillin G, is added to the medium at an initial stage of the logarithmic growth phase (OD : 0.1 - 0.4) in a concentration of 0.1 to 2.0 U/ml. Culturing is continued to an OD value of 0.3 - 0.5, and then cells are harvested and washed with the SSM medium.
• penicillin such as penicillin G
• the washed cells are resuspended in a suitable hypertonic medium such as PFM medium (pH 7.0 - 8.5) wherein 0.4M sucrose and 0.01M MgCl2 ⁇ 6H2O are added to 2 fold diluted SSM medium, and RCG medium (pH 7.0 - 8.5) consisting of 5 g/l glucose, 5 g/l casein hydrolysate, 2.5 g/l yeast extract, 3.5 g/l K2HPO4, 1.5 g/l KH2PO4, 0.41 g/l MgCl2 ⁇ 6H2O, 10 mg/l FeSO4 ⁇ 7H2O, 2 mg/l MnSO4 ⁇ (4-6)H2O, 0.9 mg/l ZnSO4 ⁇ 7H2O, 0.4 mg/l CuSO4 ⁇ 5H2O, 0.09 mg/l Na2B4O7 ⁇ 10H2O, 0.04 mg/l (NH4)6Mo7O24 ⁇ 4H2O, 30 ug/l biotin,
• lysozyme is added to a final concentration of 0.2 to 10 mg/ml, and the mixture is allowed to react at a temperature of 30 to 37°C.
• Protoplast formation proceeds with time and is monitored with an optical microscope. The period required for the conversion of most cells to protoplasts depends on the concentrations of the penicillin used for the lysozyme-sensitization and the amount of lysozyme used. The period is 3 - 24 hours under the conditions mentioned above.
• the protoplasts are prepared above have colony-forming (regenerating) ability on a suitable hypertonic agar medium.
• a suitable hypertonic agar medium a nutrient medium, a semi-synthetic medium or a synthetic medium containing various amino acids, which contains 0.3 to 0.3M sodium succinate and 0.5 to 6% polyvinyl pyrrolidone with a molecular weight of 10, 000 or 40,000 is preferably used.
• a semi-synthetic medium RCGP pH 7.2 wherein 1.4% agar is added to the RCGP agar medium is used. Regeneration is carried out at a temperature of 25 to 35°C.
• the cultivation time required for the regenration of protoplasts depends upon the strain used, and usually in 10 to 14 days colonies can be picked up.
• the efficiency of the regeneration of protoplasts on the RCGP medium also depends on the strain used, the concentrations of the penicillin added during the cultivation and the concentration of lysozyme used. The efficiency is generally 10 ⁇ 2 - 10 ⁇ 4 cells per normal cell treated with lysozyme.
• Introduction of a recombinant DNA into the protoplast is carried out by mixing the protoplast and the DNA in a hypertonic solution which protects the protoplast and by adding to the mixture polyethyleneglycol (PEG, average molecular weight: 1,540 - 6,000) or polyvinylalcohol (PVA, degree of polymerization: 500 - 1,500) and a divalent metal cation which stimulates the uptake of DNA.
• PEG polyethyleneglycol
• PVA polyvinylalcohol
• a divalent metal cation which stimulates the uptake of DNA.
• PEG and PVA can be used at a final concentration of 5 to 60% and 1 to 20%, respectively.
• Divalent metal cations such as Ca++, Mg++, Mn++, Ba++ and Sr++ are effectively used alone or in combination at a final concentration of 1 to 100 mM. Transformation is carried out satisfactorily at 0 to 25°C.
• Regeneration of the protoplast transformed with a recombinant DNA is carried out in the same way as mentioned above by spreading the protoplast on a hypertonic agar medium such as RCGP medium containing sodium succinate and polyvinyl pyrrolidone and incubating at a temperature wherein normal cells can grow, generally 25 to 35°C.
• Transformants are obtained by selecting the phenotypes derived from donor DNAs. The selection by characteristic phenotype endowed may be carried out simultaneously with regeneration on a hypertonic agar medium or may be carried out on a hypotonic agar medium after non-­selective reversion to normal cells on a hypertonic agar medium.
• transformation may be carried out by the steps described in (1) to (3) except that the cultured cells are directly treated with lysozyme without prior treatment with penicillin in step (1). In that case, transformants are obtained at an efficiency of 10 ⁇ 2 to 10 ⁇ 4 per regenerated cell.
• strains are the examples of the transformants obtained by the present invention.
• the phenotypic expression of the recombinant DNA is carried out by growing the transformant in a conventional nutrient medium. Appropriate reagents may be added to the medium according to the phenotypes expected from the gene DNA or vector DNA on the recombinant DNA.
• the thus obtained transformant is cultured in a conventional manner used in the production of amino acids by fermentation. That is, the transformant is cultured in a conventional medium containing carbon sources, nitrogen sources, inorganic materials, amino acids, vitamines, etc. under aerobic conditions, with adjustment of temperature and pH. Amino acids, thus accumulated in the medium are recovered.
• various carbohydrates such as glucose, glycerol, fructose, sucrose, maltose, mannose, starch, starch hydrolyzate and molasses, polyalcohols and various organic acids such as pyruvic acid, fumaric acid, lactic acid and acetic acid may be used.
• hydrocarbon and alcohols are employed. Blackstrap molasses is most preferably used.
• ammonia various inorganic or organic ammonium salts as ammonium chloride, ammonium sulfate, ammonium carbonate and ammonium acetate, urea, and nitrogenous organic substances such as peptone, NZ-amine, means extract, yeast extract, corn steep liquor, casein hydrolyzate, fish meal or its digested product, defatted soybean or its digested product and chrysalis hydrolyzate are appropriate.
• potassium dihydrogenphosphate dipotassium hydrogenphosphate
• ammonium sulfate ammonium chloride
• magnesium sulfate sodium chloride
• ferrous sulfate manganese sulfate and calcium carbonate
• Vitamines and amino acids required for the growth of microorganisms may not be added, provided that they are supplied with other components mentioned above.
• Culturing is carried out under aerobic conditions with shaking or aeration-agitation.
• Culturing temperature is preferably 20 to 40°C.
• the pH of the medium during culturing is maintained around neutral. Culturing is continued until a considerable amount of an amino acid is accumulated, generally for 1 to 5 days.
• accession numbers, deposition date and transfer date of the deposits under the Budapest Treaty of the International Recognition of the Deposit of Microorganisms for the purposes of Patent Procedure are as follows.
• pCG11 used as a vector plasmid was prepared from Corynebacterium glutamicum LA 103/pCG11, ATCC 39022 which is a derivative of Corynebacterium glutamicum L-22 and harbors pCG11 as follows.
• the strain was grown with shaking at 30°C in 400 ml of NB medium to an OD value of about 0.7.
• Cells were harvested and washed with TES buffer.
• the cells were suspended in 10 ml of the aforementioned lysozyme solution and allowed to react at 37°C for 2 hours.
• 2.4 ml of 5M NaCl, 0.6 ml of 0.5M EDTA (pH 8.5) and 4.4 ml of a solution consisting of 4% sodium lauryl sulfate and 0.7M NaCl were added successively.
• the mixture was stirred slowly and allowed to stand on an ice water bath for 15 hours.
• the whole lysate was centrifuged at 4°C under 69,400 x g for 60 minutes.
• the supernatant fluid was recovered and 10% (by weight) polyethyleneglycol (PEG) 6,000 (product of Nakarai Kagaku Yakuhin Co.) was added.
• PEG polyethyleneglycol
• the mixture was stirred slowly to dissolve completely and then kept on an ice water bath. After 10 hours, the mixture was subjected to centrifugation at 1,500 x g for 10 minutes to recover a pellet.
• the pellet was redissolved gently in 5 ml of TES buffer and 2.0 ml of 1.5 mg/ml ethidium bromide was added. Then, cesium chloride was added to adjust the density of the mixture to 1.580.
• the solution was centrifuged at 18°C at 105,000 g for 48 hours. After the density gradient centrifugation, a covalently-closed circular DNA was detected under UV irradiation as a high density band located in the lower part of the centrifugation tube. The band was taken out from the side of the tube with an injector to obtain a fraction containing pCG11 DNA. To remove ethidium bromide, the fraction was treated five times with an equal amount of cesium chloride saturated isopropyl alcohol solution consisting of 90% by volume isopropyl alcohol and 10% TES buffer solution. Then, the residue was dialysed against TES buffer solution to obtain pCG11 plasmid DNA.
• the chromosomal DNA of Corynebacterium glutamicum K38 FERM P-7087 was prepared by the following method:
• a seed culture was inoculated into 400 ml of SSM. Culturing was carried out with shaking at 30°C. NB medium was used as seed culture. The optical density (OD) at 660 nm was monitored with a Tokyo Koden colorimeter and penicillin G was added at an OD value of 0.2 to a concentration of 0.5 unit/ml. Culturing was continued to an OD value of about 0.6.
• Cells were harvested from the culture broth and washed with TES buffer. The cells were suspended in a lysozyme solution (pH 8.0) consisting of 25% sucrose, 0.1M NaCl, 0.05M Tris and 0.8 mg/ml lysozyme (the same lysozyme solution was used hereinafter) to make 10 ml of a suspension which was allowed to react at 37°C for 4 hours. High molecular chromosomal DNAs were isolated from the cells by the method of Saito et al .
• pCG11 used as a vector was prepared by the method described in Example 1.
• restriction enzyme BglII was added to 100 ⁇ l of the BglII reaction solution containing 3 ⁇ g of pCG11 plasmid DNA
• 5 units of BamHI was added to 100 ⁇ l of restriction enzyme BamHI reaction solution (pH 8.0) consisting of 10 mM Tris, 7 mM MgCl2, 100 mM NaCl, 2 mM mercaptoethanol P and 0.01% bovine serum albumin (the same BamHI reaction solution was used hereinafter) and containing 9 ⁇ g of the chromosomal DNA.
• BamHI reaction solution pH 8.0
• the mixtures were allowed to react at 37°C for 60 minutes and heated at 65°C for 10 minutes to stop the reactions.
• the reaction mixtures were mixed with each other.
• the above ligation mixture was provided for the following transformation.
• Corynebacterium glutamicium L-15 ATCC 31834 was used as the recipient for the transformation.
• the seed culture of L-15 strain was inoculated into NB medium and culturing was carried out with shaking at 30°C. Cells were harvested at an OD value of 0.6.
• the cells were suspended at about 109 cells/ml in RCGP medium (pH 7.6) containing 1 mg/ml lysozyme. The suspension was put in an L-­tube and stirred slowly at 30°C for 5 hours to obtain protoplasts.
• 0.5 ml of the protoplast suspension was put in a small test tube and centrifuged at 2,500 x g for 5 minutes.
• the protoplasts were resuspended in 1 ml of TSMC buffer and again subjected to centrifugation and washing.
• the washed protoplasts were resuspended in 0.1 ml of TSMC buffer solution.
• 100 ⁇ l of a mixture (1 : 1 by volume) of a two-fold concentrated TSMC buffer and the ligated DNA mixture described above was added to the protoplast suspension.
• 0.8 ml of a solution containing 20% PEG 6,000 in TSMC buffer solution was added to the mixture.
• RCGP medium pH 7.2
• RCGP medium pH 7.2
• the supernatant fluid was removed and the protoplasts were suspended in 1 ml of RCGP medium.
• 0.2 ml of the suspension was spread on RCGP agar medium containing 200 ⁇ g/ml spectinomycin and incubated at 30°C for 7 days.
• spectinomycin-resistant colonies formed on the selection plate were scraped, washed with physiological saline solution and centrifuged two times. The cells were spread on a minimal agar medium M1 containing 100 ⁇ g/ml spectinomycin and 0.5 mg/ml PFP and incubated at 30°C for 2 days. The transformants which are resistant to spectinomycin and PFP were obtained from the colonies formed.
• Plasmid DNAs were isolated from cells of these transformants.
• the plasmid pCS-CM1 recovered from one of the transformants was analyzed by digestion with various restriction endonucleases and analyzed by agarose gel electrophoresis. The analysis showed at a BamHI DNA fragment of about 9.4 Kb was inserted into the unique BglII cleavage site of pCG11 in pCS-­CM1.
• Plasmid DNAs were isolated from these transformants as mentioned above.
• a plasmid pCS-CM2 obtained from one of the transformants was digested with restriction endonucleases and analysed by agarose gel electrophoresis. The analysis showed that a BamHI DNA fragment of about 9.4 Kb was inserted into the unique BglII cleavage site of pCG11 in pCS-CM2.
• the BamHI DNA fragment of about 9.4 Kb cloned in pCS-CM1 and pCS-CM2 contains the genes encoding for chorysmate mutase and prephenate dehydratase of Corynebacterium glutamicum K38 and that the DNA fragment can confer resistance to para-fluorophenylalanine on Corynebacterium glutamicum .
• Corynebacterium glutamicum K38 (FERM P-7087, FER ⁇ BP-­454) which produces phenylalanine was transformed with pCS-CM2 as mentioned above.
• the thus obtained transformant has been deposited with the Fermentation Research Institute as Corynebacterium glutamicum K39, FERM P-7088 (FERM BP-459).
• Corynebacterium glutamicum K39, carrying pCS-CM2 was tested for L-phenylalanine production as follows.

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• 1984
  • 1984-02-16 DE DE89108172T patent/DE3486188T2/de not_active Expired - Fee Related
  • 1984-02-16 EP EP89108165A patent/EP0336452B1/fr not_active Expired - Lifetime
  • 1984-02-16 DE DE89108165T patent/DE3486229T2/de not_active Expired - Fee Related
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  • 1984-02-16 AU AU25721/84A patent/AU568340B2/en not_active Ceased
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